COPLA, a taxonomic classifier of plasmids

Background: Plasmids are mobile genetic elements, key in the dissemination of antibiotic resistance, virulence determinants and other adaptive traits in bacteria. Obtaining a robust method for plasmid classification is necessary to better understand the genetics and epidemiology of many pathogens. Until now, plasmid classification systems focused on specific traits, which limited their precision and universality. The definition of plasmid taxonomic units (PTUs), based on average nucleotide identity metrics, allows the generation of a universal plasmid classification scheme, applicable to all bacterial taxa. Here we present COPLA, a software able to assign plasmids to known and novel PTUs, based on their genomic sequence. Results: We implemented an automated pipeline able to assign a given plasmid DNA sequence to its cognate PTU, and assessed its performance using a sample of 1000 unclassified plasmids. Overall, 41% of the samples could be assigned to a previously defined PTU, a number that reached 63% in well-known taxa such as the Enterobacterales order. The remaining plasmids represent novel PTUs, indicating that a large fraction of plasmid backbones is still uncharacterized. Conclusions: COPLA is a bioinformatic tool for universal, species-independent, plasmid classification. Offered both as an automatable pipeline and an open web service, COPLA will help bacterial geneticists and clinical microbiologists to quickly classify plasmids.This work was supported by the Spanish Ministry of Science and Innovation [PID2020-117923GB-I00 to FdlC]; the Spanish Ministry of Economy, Industry and Competitiveness [DI-17-09164 to SR-S]; and USA Centers for Disease Control and Prevention [200-2019-06679 to FdlC]. The funders had no role in the design of the study, nor in the collection, analysis, and interpretation of data, nor in writing the manuscript included in this submission

Genome Sequences of 18 Salmonella enterica Serotype Hadar Strains Collected from Patients in the United States

Despite being linked to a number of recent poultry-associated outbreaks in the United States, few reference genomes are available for Salmonella enterica serotype Hadar. Here, we address this need by reporting 18 Salmonella Hadar genomes from samples collected from patients in the United States between 2014 and 2020

Failure of moxifloxacin treatment in Mycoplasma genitalium infections due to macrolide and fluoroquinolone resistance

Increasing azithromycin treatment failure in sexually transmitted Mycoplasma genitalium infection, is linked to macrolide resistance and second-line treatment relies on the fluoroquinolone, moxifloxacin. We recently detected fluoroquinolone and macrolide resistance-associated mutations in 15% and 43%, respectively, of 143 initial M. genitalium PCR-positive specimens.For a subset of 33 Western Sydney Sexual Health Centre patients, clinical information and results of sequence analysis of M. genitalium macrolide and fluoroquinolone target genes - the 23S rRNA gene, and parC and gyrA, respectively - were used to examine whether mutations were associated with treatment failure. Macrolide resistance-associated mutations correlated with microbiological (p = 0.013) and clinical (p = 0.024) treatment failure, and fluoroquinolone resistance-associated mutations with microbiological moxifloxacin treatment failure (p = 0.005). We describe the first reported cases of clinical and microbiological moxifloxacin treatment failure. Failure of first- and second-line antibiotic treatment of M. genitalium infection is occurring and likely to increase with current treatment strategies.7 page(s

Fluoroquinolone and macrolide resistance-associated mutations in Mycoplasma genitalium

Mycoplasma genitalium is a significant sexually transmitted pathogen, causing up to 25% of cases of nongonococcal urethritis in men, and it is strongly associated with cervicitis and pelvic inflammatory disease in women. Currently, the usual first-line treatment is the macrolide antibiotic azithromycin, but an increasing incidence of treatment failure over the last 5 years suggests the emergence of antibiotic resistance. The mutations responsible for macrolide resistance have been found in the 23S rRNA gene in numerous M. genitalium populations. A second-line antibiotic, the fluoroquinolone moxifloxacin, was thought to be a reliable alternative when azithromycin began to fail, but recent studies have identified mutations that may confer fluoroquinolone resistance in the genes parC and gyrA. The aim of this study was to determine the prevalence of antibiotic resistance in M. genitalium in Sydney, Australia, by detecting relevant mutations in the 23S rRNA gene, parC, and gyrA. M. genitalium-positive DNA extracts of specimens, collected from patients attending sexual health clinics in Sydney, were tested by PCR amplification and DNA sequence alignment. The 186 specimens tested included 143 initial patient specimens and 43 second, or subsequent, specimens from 24 patients. We identified known macrolide resistance-associated mutations in the 23S rRNA gene in 43% of the initial patient samples and mutations potentially associated with fluoroquinolone resistance in parC or gyrA sequences in 15% of the initial patient samples. These findings support anecdotal clinical reports of azithromycin and moxifloxacin treatment failures in Sydney. Our results indicate that further surveillance is needed, and testing and treatment protocols for M. genitalium infections may need to be reviewed.5 page(s

MALDI-TOF Mass Spectrometry for Multilocus Sequence Typing of <i>Escherichia coli</i> Reveals Diversity among Isolates Carrying <i>bla</i>_CMY-2-Like Genes

<div><p>Effective surveillance and management of pathogenic <i>Escherichia coli</i> relies on robust and reproducible typing methods such as multilocus sequence typing (MLST). Typing of <i>E</i>. <i>coli</i> by MLST enables tracking of pathogenic clones that are known to carry virulence factors or spread resistance, such as the globally-prevalent ST131 lineage. Standard MLST for <i>E</i>. <i>coli</i> requires sequencing of seven alleles, or a whole genome, and can take several days. Here, we have developed and validated a nucleic-acid-based MALDI-TOF mass spectrometry (MS) method for MLST as a rapid alternative to sequencing that requires minimal operator expertise. Identification of alleles was 99.6% concordant with sequencing. We employed MLST by MALDI-TOF MS to investigate diversity among 62 <i>E</i>. <i>coli</i> isolates from Sydney, Australia, carrying a <i>bla</i>_CMY-2-like gene on an IncI1 plasmid to determine whether any dominant clonal lineages are associated with the spread of this globally-disseminated resistance gene. Thirty-four known sequence types were identified, including lineages associated with human disease, animal and environmental sources. This suggests that the dissemination of <i>bla</i>_CMY-2-like-genes is more complex than the simple spread of successful pathogenic clones. <i>E</i>. <i>coli</i> MLST by MALDI-TOF MS, employed here for the first time, can be utilised as an automated tool for large-scale population analyses or for targeted screening for known high-risk clones in a diagnostic setting.</p></div

Examples of discrepancies between MLST by MALDI-TOF MS and sequencing.

<p>^a These alleles differ by the presence/absence of a 10,290 Da peak that is often not detected by the software but is visible upon manual inspection. Other alleles that differ by the presence/absence of this peak are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143446#pone.0143446.s005" target="_blank">S3 Table</a>.</p><p>^b Indistinguishable alleles have identical spectral patterns for all four cleavage reactions. Other indistinguishable alleles can be determined by the simulation software.</p><p>^c Workflow for discrepant alleles detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143446#pone.0143446.s001" target="_blank">S1 Fig</a>.</p><p>Examples of discrepancies between MLST by MALDI-TOF MS and sequencing.</p

Sequence types of <i>E</i>. <i>coli</i> strains carrying IncI1-<i>bla</i>_CMY-2 plasmids.

<p>^a Sequence type</p><p>^b Sequence type complex based on <a href="http://mlst.warwick.ac.uk/mlst/dbs/Ecoli" target="_blank">http://mlst.warwick.ac.uk/mlst/dbs/Ecoli</a>;–, no STC</p><p>^c One isolate has a new combination of alleles and one has a new <i>gyrB</i> variant.</p><p>Sequence types of <i>E</i>. <i>coli</i> strains carrying IncI1-<i>bla</i>_CMY-2 plasmids.</p

Pathways of gene dissemination within the Salmonella enterica plasmidome

Trabajo presentado en el International symposium Salmonella and salmonellosis, celebrado en Saint-Malo (Francia) del 20 al 22 de junio de 2022

Genome sequences of 18 salmonella enterica serotype hadar strains collected from patients in the United States

Despite being linked to a number of recent poultry-associated outbreaks in the United States, few reference genomes are available for Salmonella enterica serotype Hadar. Here, we address this need by reporting 18 Salmonella Hadar genomes from samples collected from patients in the United States between 2014 and 2020.This work was supported through the CDC

Mapa plasmídico de Salmonella enterica

Trabajo presentado en el XXVIII Congreso de la Sociedad Española de Microbiología, celebrado en modalidad virtual del 28 de junio al 02 de julio de 2021.Los genes de resistencia a los antibióticos se propagan rápidamente por transferencia genética horizontal, principalmente transportados por plásmidos. De hecho, los plásmidos parecen ser los actores más importantes en la evolución a corto plazo de patógenos bacterianos, como se ejemplifica en las enterobacterias. Por lo tanto, razonamos que será relevante identificar cuál es la importancia relativa de diferentes unidades taxonómicas plasmídicas (PTUs) en la diseminación de genes de resistencia a antibióticos en microorganismos patógenos. A través de una combinación de genómica funcional y metadatos asociados a los plásmidos, ofrecemos un diagnóstico de los plásmidos más relevantes en la diseminación de genes de virulencia y resistencia a antibióticos en Salmonella enterica, así como información sobre la ecología de los brotes multirresistentes